Brown Et Al 2016 Cleangenes
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Freshwater Crayfish 22(1):43–51, 2016 RESEARCH ARTICLE ISSN: 2076-4324 (Print), 2076-4332 (Online) https://doi.org/10.5869/fc.2016.v22-1.43 Ectosymbiotic Cleaners Induce Down-regulation of Crayfish Immune Response Genes BRYAN L. BROWN,1,* MATTHEW TURNBULL,2 JAMES SKELTON 3,5 AND ROBERT P. CREED 4 1 Department of Biological Sciences, 2105 Derring Hall, Virginia Tech, Blacksburg, VA 24061 USA. *Corresponding Author.— [email protected] 2 Department of Biological Sciences, Clemson University, Clemson, SC 29631 USA. 3 School of Forest Resources and Conservation, University of Florida , Gainesville, FL 32601 USA. 4 Department of Biology, Appalachian State University, Boone, NC 28607 USA 5 Current Address: School of Forest Resources, University of Florida, Gainesville, Florida, 32601 USA. ABSTRACT ARTICLE INFO Recent studies have revealed integral and complex relationships between hosts and their symbionts. Article History: Submitted: 02 JUL 2016 Some of these findings demonstrate that symbionts can affect the gene expression of their hosts. Accepted: 15 DEC 2016 We used a model cleaning symbiosis system of crayfish and their branchiobdellidan symbionts to Published Online: 31 DEC 2016 examine whether symbionts could affect host gene expression through indirect means, by changing the Published Print: 31 DEC 2016 interaction of the host crayfish with the environment. Previous research has shown this symbiosis to Keywords: be a complex, context dependent relationship in which outcomes can shift between mutualism, when crayfish; branchiobdellidans act as cleaners of their hosts, and parasitism, when damage to hosts’ gills through branchiobdellidan; branchiobdellidan feeding produces negative effects. These shifts are known to occur with changes in symbiosis; gene expression; symbiont densities and environmental conditions. We manipulated densities of branchiobdellidans on mutualism; host crayfish and examined the effect on expression of 3 candidate immune genes. We had 2 competing cleaning symbiosis; working hypotheses: 1) That branchiobdellidans would cause an increase expression in immune genes because of damage to the hosts’ gills; and 2) That branchiobdellidans would decrease expression of immune genes by acting as cleaners and thus alleviating environmental stress. We found that the second hypothesis was strongly supported when branchiobdellidan activity decreased expression in 2 of the 3 candidate genes. One of these genes (astacidin) is related to generalized immune defense, while the other (prophenoloxidase) is a key part of the melanization cascade that provides defense in the case of physical damage and intrusion of microbes or foreign bodies. Given that there is no known mechanism through which branchiobdellidans can directly affect host physiology through chemical or genetic interactions, we concluded that this alteration in gene expression occurred through alleviation of environmental stress by the symbionts. Such indirect effects may be common in nature and continued study using easily manipulated systems like the crayfish-branchiobdellidan symbiosis may continue to produce insights regarding the importance of symbioses in ecological systems. Copyright © 2016 by The Author(s). Published by the International Association of Astacology. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. INTRODUCTION our early Egyptian pioneers would have ever imagined. We now The more we know about symbiosis, the more we appreciate know that symbionts can exert truly astonishing effects on their the ubiquity and importance of symbiotic interactions. While the hosts. Some of these relationships are quite complicated and even study of symbiosis dates back at least to the ancient Egyptians who bizarre, like fungi and parasitic worms that literally exert “mind included intestinal symbionts in early medical texts circa 1550 control” over their insect hosts (Thomas et al. 2002; Mongkolsamrit BCE (Egerton 2014), modern science has revealed that the biology et al. 2012). Even familiar and well-publicized symbioses like the of symbionts and hosts are often far more intimately entwined than relationships between plants and their pollinators, the influences of 43 44 Freshwater Crayfish Volume 22, Number 1 mycorrhizal symbioses, and the interdependence between corals thought to occur because branchiobdellidans clean crayfish body and their endosymbiotic dinoflagellates have proven to be far surfaces, particularly the gills, through their feeding behaviors, more complicated and varied than previously thought (Rodriguez- but can shift to direct consumption of host gill tissue when Lanetty et al. 2006; Karst et al. 2008; Carstensen et al. 2016). resources are limited (Brown et al. 2002; Brown et al. 2012). Recent work even suggests that our own evolutionary history, The relationship is context-dependent and can shift between current health, and survival as a species are intimately related to mutualism, commensalism, and parasitism based on a variety our microbial symbionts (Backhed et al. 2005; Yatsunenko et al. of factors including symbiont density (Brown et al. 2012), host 2012; Le Chatelier et al. 2013; Moeller et al. 2014). ontogeny (Thomas et al. 2016), and environmental conditions (Lee et al. 2009). Both host and symbiont can exert some influence One relatively recently discovered type of host-symbiont over the relationship, with hosts using grooming to remove excess interaction is the influence of symbionts on host gene expression. branchiobdellidans (Farrell et al. 2014; Skelton et al. 2014; Skelton These effects have been documented in a wide range of host- et al. 2016), and the branchiobdellidans showing evidence of host symbiont interactions, including bacteria and protozoans (Choi et choice (Brown and Creed 2004) and selective dispersal to optimize al. 1997), plants and mycorrhizal fungi (Zouari et al. 2014), and their own fitness (Skelton et al. 2015). bacteria that produce bioluminescence in several species of marine invertebrates (Almada and Tarrant 2016). We call these types of To examine the influence of branchiobdellidan symbionts interactions “recently discovered” because, even though they on host immune function, we conducted an experiment in which have been suspected for some time, only recently has technology we manipulated branchiobdellidan density on crayfish hosts, and allowed a thorough investigation of gene expression in real time. measured the expression of 3 candidate immune genes in the host To date, the majority of described cases in which symbionts affect crayfish during a 6-day experiment. In this experiment, we created the gene expression of hosts is through the symbiont directly conditions in which there was relatively high potential for microbial interacting with host physiology, for example, the influence of gill fouling of crayfish, a phenomenon that can be harmful or even Wolbachia on wasp hosts (Kremer et al. 2012), bacterial influence lethal to crustaceans (Bauer 1998), but in crayfish this fouling can over plant flowering (Lutay et al. 2016), arbuscular mycorrhizal be ameliorated to some degree by branchiobdellidans (Lee et al. fungi affecting immune defense of grapes against nematodes (Li et 2009). We also used a fairly high density of branchiobdellidans al. 2006), or the protozoan Trypanasoma promoting susceptibility as our experimental treatment so that significant gill damage to to transmission in mammals by altering expression in several the host was also a possibility (Brown et al. 2012). We therefore host pathways (Garcia-Silva et al. 2014). These previous studies had two competing working hypotheses regarding the effect of of symbiont effects on host gene expression mostly involve branchiobdellidans on host gene expression. 1) That we would endosymbionts, usually bacteria or fungi, that affect host gene measure decreased expression of immune-related genes in the expression through direct interactions with host physiology. branchiobdellidan treatments relative to the controls because the However, it is also probable that some types of symbionts can worms were alleviating gill fouling by cleaning. 2) That we would indirectly affect gene expression of their hosts by changing measure up-regulation of immune associated genes in response to the interaction between the host and environment. One type tissue damage caused by branchiobdellidans at fairly high densities. of symbiotic interaction in which these types of indirect effects Both of these hypotheses are compared to a null hypothesis of no might be expected are cleaning symbioses in which ectosymbionts effect of branchiobdellidans on host gene expression. remove parasites or foreign material from hosts, providing a beneficial cleaning service (Limbaugh 1961; Grutter et al. 2002). METHODS In these types of interactions, cleaners rarely interact with hosts in Laboratory Experiment such a way that host physiology is directly affected, but cleaners We conducted a 6-day aquarium experiment in June of can change the way that hosts interact with their environments, 2010 in which we manipulated branchiobdellidan density on 24 and thus could be expected to alter expression of host genes that host crayfish. The presence or absence of branchiobdellidans control a host’s interaction with their environment. was the only treatment in the experiment. We manipulated We investigated the potential